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Photo: Gino Fornaciari, University of Pisa

The investigators were trying to find the reason why the face of a 2-year-old girl, mummified these last 450 years, was marked with a noticeable rash.

They did not find smallpox in their DNA analysis, as had been expected. Instead, they found a form of Hepatitis B so similar to modern strains that the researchers were unsure if there had been contamination, they now report in the open access journal PLoS Pathogens.

Their conclusions: the virus that infects about a third of all people globally at some point in their lives hasn’t changed all that much.

“This phylogenetic pattern indicates that the genotypes of HBV diversified long before the 16th century, and enables comparison of potential pathogenic similarities between modern and ancient HBV,” they report. “These results have important implications for our understanding of the emergence and evolution of this common viral pathogen.”

The 2-year-old child was disemboweled and mummified sometime in the mid-1500s, and then secreted away in the sacristy of the Basilica of Saint Domenico Maggiore in Naples. The little body was exhumed during excavations of hundreds of mummies in the sacristy, sometime between 1983 and 1985. The initial autopsy of the mummy was performed by “paleopathologists wearing sterile surgical coasts, sterile latex gloves, sterile masks, headdresses and overshoes.” The body was also stored in sealed and sterile plastic bags after the initial analyses – which concluded the debilitating facial rash on the child was probably due to smallpox.

But the researchers could find no proof of a pox virus causing the rash. Young children infected with hepatitis can exhibit a facial rash now known as Gianotti-Crosti syndrome. So they took eight samples of tissue from the distal femur, skin attached to one rib, skin attached to the frontoparietal bone, muscle from the thigh, and other skin samples. Those tissue samples were sequenced at the advanced DNA facilities at McMaster University in Ontario, with an eye toward producing mitochondrial DNA, Variola virus (smallpox), and Hepatitis B genomes.

They sequenced the unknown child’s mitochondrial DNA. And while the scientists did not succeed in finding any proof of smallpox, they did find the Hepatitis B – and a familiar form of it.

“It clusters closely with modern HBV sequences, as might be expected if it were a recent contaminant,” they write.

A reconstruction of the viral family tree was the next step. Using an HBV sample from late 1600s Korea that had previously been identified, they conducted “regressions of root-to-tip genetic distance” over time using computer models. Crunching the data using analyses involving Markov chain Monte Carlo reconstructions determined that the ancient hepatitis is very similar to the samples currently circulating among humanity currently, they concluded.

“If (the virus genome) is indeed from the 16th century, then this phylogenetic pattern indicates that the diversification of the HBV genotypes occurred prior to 1500 and that any subsequent accumulation of diversity was either lost through strong purifying selection of masked by multiple substitutions,” they conclude.

Lead author Hendrik Poinar, an evolutionary geneticist at McMaster, said the investigation shows that reconstruction of the outbreaks of the past could help understanding our modern-day sicknesses.

“These data emphasize the importance of molecular approaches to help identify the presence of key pathogens in the past, enabling us to better constrain the time they may have infected humans,” he said.

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